Rapid improvements in DNA synthesis technology promise to revolutionize traditional methods employed in virology. One of the approaches traditionally used to eliminate the functions of different regions of the viral genome makes extensive but laborious use of site-directed mutagenesis to explore the impact of small sequence variations in the genomes of virus strains. However, viral genomes, especially of RNA viruses, are relatively short, often less than 10,000 bases long, making them amenable to whole genome synthesis using currently available technology. Recently developed microfluidic chip-based technologies can perform de novo synthesis of new genomes designed to specification for only a few hundred dollars each. This permits the generation of entirely novel coding sequences or the modulation of existing sequences to a degree practically impossible with traditional cloning methods.
Such freedom of design provides tremendous power to perform large-scale redesign of DNA/RNA coding sequences to: (1) study the impact of changes in parameters such as codon bias, codon-pair bias, and RNA secondary structure on viral translation and replication efficiency; (2) perform efficient full genome scans for unknown regulatory elements and other signals necessary for successful viral reproduction; and (3) develop new biotechnologies for genetic engineering of viral strains and design of anti-viral vaccines.
As a result of the degeneracy of the genetic code, all but two amino acids in the protein coding sequence can be encoded by more than one codon. The frequencies with which such synonymous codons are used are unequal and have coevolved with the cell's translation machinery to avoid excessive use of suboptimal codons that often correspond to rare or otherwise disadvantaged tRNAs (Gustafsson et al., 2004). This results in a phenomenon termed “synonymous codon bias,” which varies greatly between evolutionarily distant species and possibly even between different tissues in the same species (Plotkin et al., 2004).
Codon optimization by recombinant methods (that is, to bring a gene's synonymous codon use into correspondence with the host cell's codon bias) has been widely used to improve cross-species expression (see, e.g., Gustafsson et al., 2004). Though the opposite objective of reducing expression by intentional introduction of suboptimal synonymous codons has not been extensively investigated, isolated reports indicate that replacement of natural codons by rare codons can reduce the level of gene expression in different organisms. See, e.g., Robinson et al., 1984; Hoekema et al., 1987; Carlini and Stephan, 2003; Zhou et al., 1999. Accordingly, the introduction of deoptimized synonymous codons into a viral genome may adversely affect protein translation and thereby provide a method for producing attenuated viruses that would be useful for making vaccines against viral diseases.
Viral Disease and Vaccines
Viruses have always been one of the main causes of death and disease in man. Unlike bacterial diseases, viral diseases are not susceptible to antibiotics and are thus difficult to treat. Accordingly, vaccination has been humankind's main and most robust defense against viruses. Today, some of the oldest and most serious viral diseases such as smallpox and poliomyelitis (polio) have been eradicated (or nearly so) by world-wide programs of immunization. However, many other old viruses such as rhinovirus and influenza virus are poorly controlled, and still create substantial problems, though these problems vary from year to year and country to country. In addition, new viruses, such as Human Immunodeficiency Virus (HIV) and Severe Acute Respiratory Syndrome (SARS) virus, regularly appear in human populations and often cause deadly pandemics. There is also potential for lethal man-made or man-altered viruses for intentional introduction as a means of warfare or terrorism.
Effective manufacture of vaccines remains an unpredictable undertaking. There are three major kinds of vaccines: subunit vaccines, inactivated (killed) vaccines, and attenuated live vaccines. For a subunit vaccine, one or several proteins from the virus (e.g., a capsid protein made using recombinant DNA technology) are used as the vaccine. Subunit vaccines produced in Escherichia coli or yeast are very safe and pose no threat of viral disease. Their efficacy, however, can be low because not all of the immunogenic viral proteins are present, and those that are present may not exist in their native conformations.
Inactivated (killed) vaccines are made by growing more-or-less wild type (wt) virus and then inactivating it, for instance, with formaldehyde (as in the Salk polio vaccine). A great deal of experimentation is required to find an inactivation treatment that kills all of the virus and yet does not damage the immunogenicity of the particle. In addition, residual safety issues remain in that the facility for growing the virus may allow virulent virus to escape or the inactivation may fail.
An attenuated live vaccine comprises a virus that has been subjected to mutations rendering it less virulent and usable for immunization. Live, attenuated viruses have many advantages as vaccines: they are often easy, fast, and cheap to manufacture; they are often easy to administer (the Sabin polio vaccine, for instance, was administered orally on sugar cubes); and sometimes the residual growth of the attenuated virus allows “herd” immunization (immunization of people in close contact with the primary patient). These advantages are particularly important in an emergency, when a vaccine is rapidly needed. The major drawback of an attenuated vaccine is that it has some significant frequency of reversion to wt virulence. For this reason, the Sabin vaccine is no longer used in the United States.
Accordingly, there remains a need for a systematic approach to generating attenuated live viruses that have practically no possibility of reversion and thus provide a fast, efficient, and safe method of manufacturing a vaccine. The present invention fulfills this need by providing a systematic approach, Synthetic Attenuated Virus Engineering (SAVE), for generating attenuated live viruses that have essentially no possibility of reversion because they contain hundreds or thousands of small defects. This method is broadly applicable to a wide range of viruses and provides an effective approach for producing a wide variety of anti-viral vaccines.